Utilizing data from independent experimental approaches and computational analyses we proposed the existence for 299 sORFs in the &lt;I&gt;S. cerevisiae&lt;/I&gt;genome, representing about 5% of annotated ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and 184 of the &lt;I&gt;S. cerevisiae&lt;/I&gt;sORFs exhibit similarity with ORFs in other organisms. In collaboration with the laboratories of Jef Boeke (The Johns Hopkins University), Ron Davis (Stanford University), and Michael Snyder (Yale University), we constructed a collection of gene-deletion mutants of 140 newly identified sORFs and integrated them into the existing deletion collection. Our laboratory undertook a comprehensive analysis of the sORF deletion strains and identified 22 sORFs required for haploid growth, growth at high temperature, growth in the presence of a non-fermentable carbon source, or growth in the presence of DNA damage and replication arrest agents. Our limited analysis of the sORFs was based on a narrow range of experimental conditions and comparison to sequences currently available in the databases. We propose that additional sORFs will be identified as the databases expand. In continuation with our ongoing research objective we found that three of the newly identified sORFs have been identified as kinetochore components in global mass spectrometric studies. This result indicates that these sORFs likely have a role in kinetochore function and suggests the potential for the role of additional sORFs in kinetochore function and genome stability. We will analyze the sORF deletion strains for chromosome loss and defects in checkpoint function, and perform secondary genetic screens to further define the molecular role of the sORFs. In efforts to define other roles for sORFs, we have several ongoing collaborations to examine if the sORF deletion strains have phenotypes related to defects in cell cycle progression, nuclear transport, gene silencing and transcriptional regulation. We have also generated epitope tagged sORF strains and plasmids overexpressing the sORFs for public use. Our studies with sORFs will identify and establish the role of sORFs in diverse biological pathways. We propose that additional sORFs will be identified as the databases expand.. We will utilize computational, biochemical, and genomic approaches to validate the presence of sORFs and understand their biological function.